JP6750443B2 - Platelet aggregation analysis method, platelet aggregation analysis device, platelet aggregation analysis program, and platelet aggregation analysis system - Google Patents

Description

The present invention relates to a platelet aggregation ability analysis method, a platelet aggregation ability analysis device, a platelet aggregation ability analysis program, and a platelet aggregation ability analysis system.

Anticoagulant therapy and antiplatelet therapy are indispensable treatments for the prevention of thrombosis, and their usefulness has been shown by large-scale clinical trials.
However, antiplatelet therapy may be less effective in reducing arterial thrombosis than the effect of anticoagulant therapy in reducing cerebral infarction.

In anticoagulation therapy, the prothrombin time international standard ratio (PT-INR) and thrombotest are used to monitor the drug efficacy appropriately for each patient, while in antiplatelet therapy, the sensitivity and efficacy of antiplatelet drugs. It is considered that one of the causes is that the monitoring method has not been established yet, although it has been reported that there is a considerable individual difference in the persistence of.

Therefore, even in antiplatelet therapy, if the drug effect can be appropriately monitored by an easy-to-understand method, it will be a means for exploring an appropriate method of using the drug in each case, and improvement of the therapeutic effect can be expected.

Here, the most basic functions of platelets are adhesion and aggregation. The platelet aggregation test method for determining the adhesion state of platelets is the most widely used method for quantitatively measuring this basic function. Specifically, there are 1) transparency method, 2) impedance method, 3) particle calculation method, etc. In these test methods, it is possible to know the details of the decrease and enhancement of the blood microscopic function depending on the type and concentration of the aggregation-inducing substance used. In particular, the transmittance method is commonly used as a daily inspection.

1) The permeability method is a method of quantifying platelet aggregation over time by utilizing the fact that the transparency of PRP increases as platelet aggregation in platelet-rich plasma (PRP) increases by adding a platelet stimulating substance.

The problem with the transparency method is that
(i) Since the separation of plasma is essential, sample processing until measurement is complicated, the amount of PRP obtained by each centrifugation condition is different, the recovery rate of platelets is not constant, and during the PRP separation operation , Dense platelets precipitate with red blood cells, their aggregation behavior is not observable,
(ii) Since the strength of platelet aggregation (aggregation rate) depends to some extent on the number of platelets in PRP, when the number of platelets is 100,000/μl or less, the difference in absorbance before and after aggregation is small and a slight change. Cannot be detected,
(iii) Testing with turbid plasma such as whole blood and chylous plasma is not possible,
(iv) poor correlation between formation of platelet aggregates and light transmission,
Etc.

2) Impedance method is a method of detecting platelet aggregation as a change in electrical resistance between electrodes.The ability to aggregate platelets in whole blood can be observed.Because there is no centrifugation operation, the aggregation behavior of all platelets can be determined. This is an observable method.

The problem with the impedance method is that
(v) The initial decrease in electrical resistance is due to the presence of red blood cells between the electrodes, making it difficult to determine the initial state of platelet aggregation.
(vi) The aggregation pattern is not stable as compared with the permeability method,
Etc.

3) The particle counting method is a method in which the number of single platelets that are not involved in the formation of platelet aggregates or aggregates is calculated with a Coulter counter and the behavior of aggregation is known.

The problem with the particle counting method is that
(vii) The procedure is complicated,
(viii) unable to record changes over time,
(ix) Platelet lysis due to aggregation-inducing substance is erroneously calculated as a decrease in single platelets,
Etc.

On the other hand, in recent years, a method of performing dielectric measurement in the blood coagulation process has been devised as a method capable of easily and accurately evaluating blood coagulation ability measurement (Patent Document 1).
This method is a method in which a capacitor-like sample portion composed of a pair of electrodes and the like is filled with blood, and an AC electric field is applied to the sample portion to measure the change in the dielectric constant due to the coagulation process of blood.

Here, the blood sample was collected from a vein using citric acid as an anticoagulant, and the anticoagulant action of citric acid was released by adding an aqueous calcium chloride solution immediately before the start of the measurement, and the blood coagulation reaction. To proceed. By analyzing the data thus obtained according to a predetermined algorithm, it is possible to obtain parameters relating to blood coagulation such as blood coagulation time.

Further, research on the dielectric measurement of the blood coagulation process is further advanced, and based on the change occurring in the complex dielectric constant spectrum measured in the blood coagulation process by adding a substance that activates or inactivates platelets to the blood. , A blood coagulation system analysis method for acquiring information on the blood coagulation ability has been developed (Patent Document 2).

In this blood coagulation system analysis method, when a platelet activating substance is used, the coagulation ability of platelets contained in the blood in an inactive state is based on the change in the complex permittivity spectrum associated with platelet activation. You can get information about. Further, when a platelet inactivating substance is used, it is possible to obtain information on the coagulability of platelets contained in the blood in an active state, based on the change in the complex dielectric constant spectrum that accompanies platelet inactivation. it can.

JP, 2010-181400, A JP2012-194087A

However, in the method of performing the dielectric measurement of the blood coagulation process and the method of analyzing the blood coagulation system, a sample (whole blood) having a normal coagulation ability is used, and the width Δts (reference value) of the reference blood coagulation time is reduced in advance. ) Had to be set.

Further, when the coagulation reaction proceeds, there is a problem that the measurement time is long because an accelerating reagent is not added. This is because when the accelerating reagent is added, the difference due to the platelet function becomes invisible.

Therefore, the inventors of the present application have conducted extensive studies to provide a simple and fast method for measuring platelet aggregation ability.
Then, a small amount of calcium chloride aqueous solution and a platelet-inducing substance were added to whole blood, and after stirring for a certain period of time, a change in the dielectric constant measured in the process of non-disturbed coagulation (natural blood coagulation) and non-platelet-inducing The inventors have found that there is a difference in the change in dielectric constant when a substance is added, and have completed the present technology.

That is, the present technology is
A step of adding a platelet-inducing substance and a calcium salt to the platelet-containing sample,
Provided is a method of analyzing platelet aggregation ability, which comprises a step of stirring the platelet-containing sample and a step of acquiring measurement data of electrical characteristics and/or viscoelasticity of the platelet-containing sample.
The measurement data of the electrical characteristics is preferably the dielectric constant of the platelet-containing sample.
Moreover, the measurement data of the viscoelasticity may be measurement data of a platelet-containing sample by a rheometer.
The platelet aggregation ability analysis method of the present technology includes measurement data of electrical characteristics and/or viscoelasticity of the platelet-containing sample, and electrical characteristics and/or viscoelasticity obtained from the platelet-containing sample to which the platelet-inducing substance is not added. The step of analyzing the platelet aggregating ability based on the measurement data of can be further included.
The platelet-containing sample may be blood or plasma.
Further, the blood or plasma may be collected from a subject who has been administered an antiplatelet aggregating drug and/or an anticoagulant drug.

This technology is
A biological sample holding unit for holding a platelet-containing sample,
A drug supply unit that supplies a platelet-inducing substance and/or a calcium salt to the platelet-containing sample,
A stirring mechanism for stirring the platelet-containing sample,
There is also provided a platelet aggregating ability analyzer including a measurement unit that measures the electrical characteristics of the platelet-containing sample.

In addition, this technology
Electrical properties and/or viscoelasticity measurement data obtained from a platelet-containing sample to which a platelet-inducing substance and a calcium salt have been added, and electrical properties obtained from a platelet-containing sample to which the platelet-inducing substance has not been added, and PROBLEM TO BE SOLVED: To provide a program for analyzing platelet aggregation ability, which causes a computer to analyze the platelet aggregation ability based on the measurement data of viscoelasticity.
In the analysis, measurement data of electrical characteristics and/or viscoelasticity obtained from the platelet-containing sample to which the platelet-inducing substance and calcium salt were added, and obtained from the platelet-containing sample to which the platelet-inducing substance was not added The measured data of the electrical characteristics and/or viscoelasticity may be compared.

Furthermore, this technology is
A biological sample holding unit for holding a platelet-containing sample,
A drug supply unit that supplies a platelet-inducing substance and/or a calcium salt to the platelet-containing sample,
A stirring mechanism for stirring the platelet-containing sample,
A measurement unit for measuring the electrical characteristics of the platelet-containing sample,
Electrical data and/or viscoelasticity measurement data obtained from the platelet-containing sample to which the platelet-inducing substance and calcium salt were added, and electrical properties obtained from the platelet-containing sample to which the platelet-inducing substance was not added And/or a platelet aggregability analysis apparatus incorporating a computer having a program for analyzing platelet aggregability, which causes a computer to analyze the platelet aggregability based on the measurement data of viscoelasticity, and the result of the analysis It is possible to provide a platelet aggregation analysis system that includes a display device that displays.

It is a figure which shows the measurement flow in the platelet aggregation capability analysis method. It is a figure which shows the outline of a structure of a platelet aggregation analyzer. It is a figure which shows the analysis flow performed by the program for platelet aggregation analysis. It is a figure which shows the outline of a structure of a platelet aggregation analysis system. It is a graph which shows the measurement result of the sample by impedance aggregation measurement. It is a graph which shows the measurement result of the sample by a dielectric constant. It is a graph which shows the example of the analysis result based on the measurement result by a dielectric constant. It is a graph which shows the example of the analysis result based on the measurement result by a dielectric constant. It is a graph which shows the correlation of the measurement data by dielectric constant, and the measurement data parameter (AUC(U)) by impedance aggregation measurement. It is a graph which shows the correlation of the measurement data by a dielectric constant, and the measurement data parameter (Aggregation(AU)) by impedance aggregation measurement. It is a graph which shows the dielectric clot intensity|strength of a sample which added a platelet-inducing substance, and a control. It is a graph which shows the correlation of the data of dielectric clot intensity based on a dielectric constant, and the measurement data parameter (AUC(U)) by impedance cohesion measurement. It is a graph which shows the correlation of the data of the dielectric clot strength based on a dielectric constant, and the measurement data parameter (Aggregation(AU)) by impedance coagulation measurement.

Hereinafter, a suitable mode for carrying out the present technology will be described. It should be noted that the embodiments described below show typical embodiments of the present technology, and the scope of the present technology is not narrowly interpreted by this. The description will be given in the following order.
1. Platelet aggregation analysis method 2. Platelet aggregation ability analyzer 3. Program for analyzing platelet aggregation ability 4. Platelet aggregation analysis system 5. Example (1) Method (2) Result (3) Analysis example 1
(4) Analysis example 2
6. Summary

1. Platelet Aggregation Ability Analysis Method The platelet aggregation ability analysis method of the present technology is
Step A: a step of adding a platelet-inducing substance and a calcium salt to a platelet-containing sample,
Step B: a step of stirring the platelet-containing sample, and step C: a step of acquiring measurement data of electrical characteristics and/or viscoelasticity of the platelet-containing sample.
In the platelet-containing sample to which the platelet-inducing substance and the calcium salt have been added in step A, the platelets activated by the platelet-inducing substance aggregate by the stirring in step B. In step C, the platelets reacted in step B do not contribute to the response, and the unreacted platelets respond in the process of blood coagulation reaction.

The platelet-containing sample to be analyzed by the present technology is not particularly limited, and examples thereof include human or mammalian blood (whole blood), plasma, artificial blood, and the like. It may also be a sample collected from a subject who has been administered an antiplatelet aggregating drug, an anticoagulant drug, or both an antiplatelet aggregating drug and an anticoagulant drug.

The platelet-inducing substance used in step A is a substance that causes or suppresses platelet aggregation. Platelet-inducing substances include, for example, collagen (COL), epinephrine, ristocetin, thrombin, thromboxane A2 (TAX2), thrombin receptor activating protein (TRAP), adenosine diphosphate (ADP), arachidonic acid (AA), serotonin. , Adrenaline and noradrenaline.

The concentration of the platelet-inducing substance added (final concentration when added to the sample) is, for example, thrombin receptor activating protein (TRAP), preferably 1 μM or more and 100 μM or less, more preferably 3 μM or more and 80 μM or less, and further preferably Is 5.4 μM or more and 62 μM or less.
If it is adenosine diphosphate (ADP), it is preferably 0.1 μM or more and 100 μM or less, more preferably 0.5 μM or more and 30 μM or less, and further preferably 1.0 μM or more and 12.5 μM or less.
In the case of arachidonic acid (AA), it is preferably 0.01 mM or more and 10 mM or less, more preferably 0.05 mM or more and 5.0 mM or less, and further preferably 0.08 mM or more and 1.0 mM or less.

The calcium salt (calcium chloride or the like) used in the step A is a substance for releasing the anticoagulant action of citric acid added to the sample at the time of blood sampling, by the contained calcium ions.
The concentration of calcium chloride added (concentration of reagent of calcium chloride) is not particularly limited as long as it has a platelet coagulation promoting effect, but preferably 150 mM or more and 250 mM or less, more preferably 170 mM or more and 230 mM or less, further preferably 185 mM or more and 215 mM. It is the following.

In step A, the timing of adding the platelet-inducing substance and the calcium salt to the platelet-containing sample is not particularly limited, but they can be added simultaneously in the present technology. Then, in step B, the platelet-containing sample, the platelet-inducing substance, and the calcium salt are stirred, and then in step C, data on the platelet coagulation ability is acquired.

The stirring condition in the step B is not particularly limited as long as it does not inhibit the platelet coagulation reaction of the platelet-containing sample, and stirring is performed at 37° C., for example. The stirring speed and strength are not particularly limited, but it is preferable to study in advance depending on the stirring method and the type of the inducer. The stirring method is not particularly limited, and examples thereof include suction/discharge with a pipette and use of a stirrer. The stirring time was found to be 5 minutes as a guideline for the platelets to react with the platelet-inducing substance and aggregate, as a result of the study in the present technology, but it is not particularly limited, and is preferably 3 to 6 minutes. .. It is shortened or extended depending on the stirring method and the type of inducer.

In step C, the electrical properties and/or viscoelasticity of the platelet-containing sample are measured.
Here, examples of the electrical characteristics include dielectric constant, impedance, admittance, capacitance, conductance, conductivity, phase angle, and the like. If it is a dielectric constant, the measuring method of the solidification process described in Japanese Patent No. 5691168 and Japanese Patent No. 5768422 may be followed.

The viscoelasticity can be measured with a rheometer. Examples of rheometers include rotation thromboelastometry, thromboelastography, and ReoRox™. As a commercially available device, a thromboelastography (TEG (registered trademark)) blood coagulation analyzer (Hemonetics), a rotation thromboelastometry (ROTEM (registered trademark)) blood coagulation analyzer (TEM group, Basel, Switzerland), etc. Can be mentioned.

As a control at the time of measuring the platelet aggregation ability, for example, a platelet-containing sample to which a calcium salt is added without adding a platelet-inducing substance may be used.
The platelet aggregation ability can be analyzed by comparing the measurement data of the platelet-containing sample with the measurement data obtained from the platelet-containing sample to which the control platelet-inducing substance is not added. The comparison can be performed, for example, by calculating the difference between the data, the ratio, and the difference between the areas of the measurement waveforms.

As described above, there are various platelet-inducing substances such as TRAP, ADP, AA, etc., but they have different functions in the coagulation system.
Therefore, a plurality of identical platelet-containing samples are prepared, a plurality of platelet-inducing substances are selected, different platelet-inducing substances and calcium salts are added to each of the platelet-containing samples, and the mixture is stirred to obtain electrical characteristics and/or viscoelasticity. By measuring and comparing the data, it becomes possible to consider the platelet coagulation ability corresponding to the platelet-inducing substance. Furthermore, it is also possible to compare the measurement results of a control in which physiological saline, a buffer solution or the like, which has little effect on platelets, is used instead of the platelet-inducing substance.

The platelet aggregation ability analysis method of the present technology can be carried out, for example, according to the measurement flowchart shown in FIG. 1.
First, blood is collected from the subject (S101). A platelet-inducing substance and a calcium salt are added to the whole blood (S102). This operation can be performed in one step. After the addition, whole blood is stirred under warm conditions (S103). The stirring time is, for example, 5 minutes.
Next, blood coagulation ability is measured (S104). The measurement can be performed without stirring. This step is a non-disturbing coagulation ability measurement in which a natural blood coagulation process is measured without adding an accelerating reagent or the like. The blood coagulation ability may be measured over time, or the time from the start of measurement may be designated to measure the coagulation ability at that time.
Finally, the obtained measurement data is analyzed to calculate the platelet contribution part in blood coagulation (S105).

2. Platelet aggregability analyzer
A biological sample holding unit for holding a platelet-containing sample,
A drug supply unit that supplies a platelet-inducing substance and/or a calcium salt to the platelet-containing sample,
A stirring mechanism for stirring the platelet-containing sample,
And a measurement unit that measures the electrical characteristics of the platelet-containing sample.

The outline of the configuration of the platelet aggregation analyzer is shown in FIG.
The biological sample holding unit 1 holds the platelet-containing sample supplied from the biological sample supply unit 2 and agitates the platelet-inducing substance and the calcium salt supplied from the drug supply unit 3.

The stirring mechanism 6 operates so as to stir the platelet-containing sample, the platelet-inducing substance, and the calcium salt supplied to the biological sample holder 1. The stirring method is not particularly limited, and for example, stirring with an electric pipette, stirring with a stirring bar, and a device with a built-in stirring function can be performed.
Stirring is performed for a certain period of time to react the platelets.

The temperature control unit 4 and the time control unit 5 control the conditions inside the biological sample holding unit. For example, the temperature control unit 4 controls the platelet-containing sample to be maintained at a constant temperature. The time control unit 5 controls the time for which the sample containing platelets is held in the biological sample holding unit 1, the stirring time, and the like.
The drive mechanism 7 performs the operation related to the biological sample holding unit 1, such as the driving operation of the temperature control unit 4 and the stirring mechanism 6, the liquid feeding of the platelet-containing sample, and the like.

The measurement unit 9 adds a platelet-inducing substance and/or a calcium salt to the platelet-containing sample, stirs it, and then measures the dielectric constant. A rheometer may be used as the measuring unit 9.
The measurement condition control unit 9 sets and adjusts temperature conditions and measurement time conditions suitable for the measurement method. Further, the measurement condition control unit 9 controls the frequency used for the measurement, the measurement interval, and the like when the dielectric constant is measured by the measurement unit 8.
The accuracy management unit 10 manages data so as not to cause a difference between measurements in the measurement unit 8 and a background variation, and monitors the state of each unit of the apparatus.

The analysis unit 11 analyzes the platelet contributing part in blood coagulation based on the measurement data based on the dielectric constant or viscoelasticity. The analysis can be performed using a program for analyzing platelet aggregation ability described below.
The analysis unit 11 may further include an output control unit that outputs the analysis result, a display device that displays the analysis result, a storage unit that stores the measurement data and the analysis result, and the like.

3. Platelet aggregation analysis program The platelet aggregation analysis program used in this technology is
Measurement data of electrical properties and/or viscoelasticity obtained from a platelet-containing sample to which a platelet-inducing substance and a calcium salt are added,
A computer is made to analyze the platelet aggregation ability based on the electrical characteristics and/or viscoelasticity measurement data acquired from the platelet-containing sample to which the platelet-inducing substance is not added.
The program is provided on a storage medium.

The platelet-containing sample to which the platelet-inducing substance is not added can be used as a control. For example, a sample to which a calcium salt is added, a sample to which physiological saline or a buffer solution is added can serve as a control. By setting a control, the platelet contribution part can be calculated by comparing and examining the difference in data caused by the presence or absence of addition of the platelet-inducing substance.

FIG. 3 shows a flowchart of the analysis performed by the program.
Feature points are extracted from the data of the measurement result (S301) obtained by the measuring unit 9 of the platelet aggregation analyzer (S302). The characteristic point is, for example, a point at which the measurement results of the control and the sample have started to be stable, a point at which the platelet coagulation time is determined, and the like. The characteristic point can be, for example, the specific time that has elapsed after the platelet-inducing substance and/or calcium salt was added to the platelet-containing sample and stirred.

At the characteristic point, the difference between the measured values of the platelet-containing sample to which the platelet-inducing substance and the calcium salt have been added and the platelet-containing sample to which the calcium salt has been added is calculated (S303), and the result is output.
The magnitude of the difference is taken as the effect of platelet function (S304). That is, it is determined that the larger the difference, the higher the platelet function (aggregation ability) in the platelet-containing sample to which the platelet-inducing substance and the calcium salt are added.

4. Platelet Aggregation Ability Analysis System An outline of the platelet aggregation ability analysis system of the present technology is shown in FIG.
The platelet aggregation analysis system 40 includes the platelet aggregation analysis device 41 and a display device 42 that displays the analysis result of the platelet aggregation analysis.
A computer equipped with the platelet aggregation analysis program may be incorporated in the platelet aggregation analysis device 41.
As the display device 42, a display, a printout device, or the like installed in the computer can be used.

The platelet aggregating ability analyzer 41 may be configured to analyze not only the platelet aggregating ability of the platelet-containing sample but also other blood coagulation system measurement items, and may also serve as a blood coagulation system analyzer.
When the platelet aggregation analyzer 41 is used as a blood coagulation system analyzer based on the dielectric constant, the measurement items include blood coagulation (coagulation), fibrin formation, fibrin clot formation, clot formation, red blood cell formation, and blood measurement. Examples include aggregation, erythrocyte sedimentation (erythrocyte sedimentation), clot contraction (regression), hemolysis, fibrinolysis and the like. When analyzing these items, a program for analyzing these items is used instead of the program for analyzing platelet aggregation ability.

The display device 42 may include a warning unit. The warning unit presets a range of normal values in each blood state change, and issues a warning when the analysis result of the sample is outside the range of normal values. The warning method is not particularly limited, and the warning can be given by, for example, a display or voice.

5. Example (1) Method Venous blood collection of a healthy person was performed by a usual method using a commercially available vacuum blood collection tube containing citric acid as an anticoagulant. The first one was discarded without use and the blood collected thereafter was used for the following experiments. In addition, after blood collection, it was used after being left at room temperature for about 30 minutes.
For the measurement of the dielectric constant, the blood coagulation system analyzers (hereinafter referred to as "dielectric coagulometer β machine") described in Japanese Patent No. 5691168 and Japanese Patent No. 5768422 were used. A platelet function analyzer Multiplate was used for impedance aggregation measurement.

First, 200 ul of whole blood was added to the cartridge for exclusive use of the dielectric coagulometer containing a platelet-inducing substance and an aqueous calcium solution (0.215M Ca), and stirred at 37°C for 5 minutes. Further, as a control, a system in which physiological saline was added instead of the platelet-inducing substance was also prepared, and stirred in the same manner.
Here, adenosine diphosphate (ADP), arachidonic acid (AA), and collagen were used as the platelet-inducing substance.
The number of specimens was N=3.

For these sample bloods, the blood coagulation ability was measured by the dielectric constant using a dielectric coagulometer β machine. In addition, impedance aggregation measurement was performed using a platelet function analyzer Multiplate, and ADPtest, ASPItest, and COLtest were performed. The measurement temperature was 37°C.
In order to confirm the reproducibility by the above method, a similar experiment by a healthy person other than the above was also performed several times.

(2) Results FIG. 5-1 shows the measurement results of Multiplate, which is a conventional platelet aggregation analyzer.
Figure 5-2 shows the measurement results of the dielectric core glomometer β machine using the dielectric constant at 10MHz.
Comparing the time variation of the expected results with the dielectric core gromometer β machine after adding physiological saline (control) and after adding the platelet-inducing substance, it was found that the amplitude changes when the platelet-inducing substance is added. ..
Since this result correlates with the change in the measurement result of Multiplate, it was shown that the agglutination reaction of platelets can be measured even by the dielectric core glomometer β machine.

(3) Analysis example 1
An example of the analysis method will be shown based on the measurement results based on the dielectric constant shown in FIG.
The study time was 20 minutes when the measured waveform of the permittivity was sufficiently stable (Fig. 6-1). The value obtained by subtracting 1 from the standard value (minimum value standard) at that time was taken as 100 times (correction to prevent the value from becoming small), and this value was defined as CF (Fig. 6-2).
The examination time is not limited to 20 minutes, and can be shortened to 15 minutes or 10 minutes if the change in permittivity is stable after the solidification time CT is determined. However, for specimens with extended CT, it was considered necessary to change the examination time rules accordingly. Therefore, the examination time may be 20 minutes or more.
It is also possible to decide (dynamically) relative to CT without fixing the examination time, for example, CT + X minutes, CT × Y minutes, or a more complex function including CT. You can

Further, as shown in FIG. 6-2, when CF of physiological saline (NaCl, control) is used as a standard, the decrease in CF due to the addition of a platelet-inducing substance is a part reflected by platelet function, particularly aggregating ability ( It is assumed that the blood glucose concentration will be close to the standard value when the platelet-contribution part) and the platelet aggregation function are decreased or when a platelet function inhibitor is taken.
For example, in patients taking clopidogrel, which is an ADP pathway inhibitor, the difference from the standard value decreases even when ADP is added.

Furthermore, it was confirmed that the CF value has a high correlation with the blood concentration-time curve area (AUC(U)) and the aggregation unit (Aggregation(AU)), which are parameters obtained from the Multiplate (FIG. 7-1, Figure 7-2). In addition, the transition of the correlation coefficient when the examination time is changed (t=5min, 10min, 15min, 20min) is shown in Tables 1 and 2 below. It can be seen from Tables 1 and 2 that if the examination time is earlier than the CT determination (t=5 min), the correlation becomes poor and it becomes difficult to capture the platelet contributing part.

(4) Analysis example 2
An example of another analysis method will be shown based on the measurement result based on the dielectric constant shown in FIG.
Dielectric clot strength (hereinafter referred to as "DCS"), which is a specific parameter for blood coagulation ability measurement by dielectric constant, was examined.
DCS is a parameter that can be calculated based on the amount of decrease from the peak of measurement data over time measured at a specific frequency.
Specifically, for example, when the time when the change in the dielectric constant at a frequency of 10 MHz is near the minimum value is defined as the clot time (hereinafter, referred to as “CT”), DCS is defined as the dielectric constant at the CT time. Then, it is calculated from the difference in permittivity when the solidification is completed (end point). Various DCS parameters can be calculated by changing the setting of the end point. Here, the end point was set to a point with a maximum gradient of 10% in the gradient of the change in permittivity after CT determination of 10 MHz.

FIG. 8 shows a graph of DCS of a sample to which calcium chloride and a platelet-inducing substance were added and DCS of a control to which calcium chloride and sodium chloride (physiological saline) were added.
Since the graph of FIG. 8 shows the same changes as those of the graph of FIG. 6-2, it was found that the information reflected by the platelet function, particularly the aggregating ability (platelet contributing part) can be obtained by calculating DCS. ..
Moreover, it was confirmed that DCS has a high correlation with AUC(U) and Aggregation(AU), which are parameters obtained from Multiplate (FIGS. 9-1 and 9-2).
As mentioned above, DCS is a parameter that can be calculated based on the amount of decrease from the peak of the measured data over time measured at a specific frequency (for example, 1 MHz). Etc. may be used or calculated.

6. Summary The present technology can measure and analyze the platelet aggregation ability of whole blood, which is a more excellent sample, as a clinical test in which thrombosis is considered.
Further, conventionally, when measuring platelet aggregating ability and blood coagulating ability, separate measuring devices were required because different measuring techniques were used, respectively. The measurement for the factor can be performed with the same device.
For example, if a plurality of measurement units capable of simultaneous measurement are installed in the device of the present technology, a control sodium salt-added sample, and a sodium salt- and platelet-inducing substance-added sample are measured to examine the platelet aggregation ability, and at the same time, The blood coagulation ability can be examined by measuring a sample containing an extrinsic/intrinsic coagulation promoter. With such an apparatus, it becomes possible to investigate the interrelationship between the platelet aggregation ability and the blood coagulation ability.

Furthermore, the present technology is useful for monitoring the administration of antiplatelet aggregating agents and/or anticoagulants to patients, monitoring the blood condition of patients during surgery, and the like.
For example, when only platelets are activated by a platelet-inducing substance, the DCS value becomes lower than that in the control (physiological saline). This is a change due to platelet contribution, and indicates that the lower DCS is due to the addition of the platelet-inducing substance, the higher the platelet function. In addition, if the DCS has a height (high value) equal to or close to that of the control despite the addition of the platelet-inducing substance, it can be inferred that the platelet function may be decreased or the antiplatelet drug may be taken. ..

Note that the present technology can also have the following configurations;
[1] A step of adding a platelet-inducing substance and a calcium salt to a platelet-containing sample,
A method for analyzing platelet aggregation, comprising: agitating the platelet-containing sample; and acquiring measurement data of electrical characteristics and/or viscoelasticity of the platelet-containing sample.
[2] The method for analyzing platelet aggregation ability according to [1], wherein the measurement data of the electrical characteristics is the dielectric constant of a platelet-containing sample.
[3] The platelet aggregation ability analysis method according to [1], wherein the measurement data of viscoelasticity is measurement data of a platelet-containing sample by a rheometer.
[4] Platelets based on measurement data of electrical characteristics and/or viscoelasticity of the platelet-containing sample and measurement data of electrical characteristics and/or viscoelasticity obtained from the platelet-containing sample to which the platelet-inducing substance is not added The method for analyzing platelet aggregation ability according to [1] or [2], which comprises a step of analyzing aggregation ability.
[5] The method for analyzing platelet aggregation ability according to any one of [1] to [4], wherein the platelet-containing sample is blood or plasma.
[6] The platelet aggregation ability analysis method according to [5], wherein the blood or plasma is collected from a subject administered with an antiplatelet aggregation drug and/or an anticoagulant drug.
[7] A biological sample holding unit that holds a platelet-containing sample,
A drug supply unit that supplies a platelet-inducing substance and/or a calcium salt to the platelet-containing sample,
A stirring mechanism for stirring the platelet-containing sample,
A platelet aggregation analysis apparatus, comprising: a measuring unit that measures the electrical characteristics of the platelet-containing sample.
[8] Measurement data of electrical properties and/or viscoelasticity obtained from a platelet-containing sample to which a platelet-inducing substance and a calcium salt are added, and electricity obtained from a platelet-containing sample to which the above-mentioned platelet-inducing substance is not added A program for analyzing platelet aggregation, which causes a computer to analyze the platelet aggregation based on the physical properties and/or measurement data of viscoelasticity.
[9] The analysis is based on measurement data of electrical characteristics and/or viscoelasticity obtained from a platelet-containing sample to which a platelet-inducing substance and a calcium salt are added, and a platelet-containing sample to which the platelet-inducing substance is not added. The program for analyzing platelet aggregation ability according to [8], which comprises comparing the obtained measurement data of electrical properties and/or viscoelasticity.
[10] A biological sample holding unit that holds a platelet-containing sample,
A drug supply unit that supplies a platelet-inducing substance and/or a calcium salt to the platelet-containing sample,
A stirring mechanism for stirring the platelet-containing sample,
A measurement unit for measuring the electrical characteristics of the platelet-containing sample,
Electrical characteristics and/or viscoelasticity measurement data obtained from a platelet-containing sample to which the platelet-inducing substance and calcium salt are added, and electrical characteristics obtained from a platelet-containing sample to which the platelet-inducing substance is not added And/or a platelet aggregating ability analysis device incorporating a computer having a program for analyzing a platelet aggregating ability, which causes a computer to analyze the platelet aggregating ability based on measurement data of viscoelasticity, and a result of the analysis A platelet aggregation analysis system comprising a display device for displaying.

1 biological sample holding unit 2 biological sample supply unit 3 drug supply unit 4 temperature control unit 5 time control unit 6 stirring mechanism 7 drive mechanism 8 measurement unit 9 measurement condition control unit 10 accuracy management unit 11 analysis unit 40 platelet aggregation analysis system 41 Platelet aggregation analysis device 42 Display device

Claims (9)

A step of adding a platelet-inducing substance and a calcium salt to the platelet-containing sample,
Stirring the platelet-containing sample to aggregate platelets activated by the platelet-inducing substance , and
After the step , a step of acquiring measurement data of electrical characteristics and/or viscoelasticity of the platelet-containing sample in a non-stirred state ,
From the measurement data of the electrical properties and/or viscoelasticity of the platelet-containing sample, and from the platelet-containing sample to which at least one selected from the group consisting of calcium salt, physiological saline and buffer solution without adding the platelet-inducing substance A method for analyzing platelet aggregation, comprising a step of analyzing platelet aggregation based on the acquired electrical characteristics and/or measurement data of viscoelasticity . The platelet aggregation ability analysis method according to claim 1, wherein the measurement data of the electrical characteristics is a dielectric constant of a platelet-containing sample. The platelet aggregation ability analysis method according to claim 1, wherein the measurement data of the viscoelasticity is measurement data of a sample containing platelets by a rheometer. The platelet aggregation ability analysis method according to claim 1, wherein the platelet-containing sample is blood or plasma. The platelet aggregation ability analysis method according to claim 4, wherein the blood or plasma is collected from a subject who has been administered an antiplatelet aggregating agent and/or an anticoagulant agent. A biological sample holding unit for holding a platelet-containing sample,
A drug supply unit that supplies a platelet-inducing substance and/or a calcium salt to the platelet-containing sample,
A stirring mechanism for stirring the platelet-containing sample to aggregate platelets activated by the platelet-inducing substance ,
A measurement unit for measuring the electrical characteristics of the platelet-containing sample in a non-stirred state ;
A time control unit for controlling the stirring time for stirring the platelet-containing sample,
A drive mechanism for controlling the drive operation of the stirring mechanism,
An analyzer for analyzing a platelet contributing part in blood coagulation based on the electrical characteristics measured by the measuring part . A platelet- inducing substance and a calcium salt are added to a platelet-containing sample and stirred, and after stirring, measurement data of electrical characteristics and/or viscoelasticity obtained from the platelet-containing sample and the platelet-inducing substance in the platelet-containing sample. added which do rather than calcium salts, are stirred at least one is added from among the group consisting of saline and buffer, after stirring, the platelet electrical characteristics obtained from a sample containing and / or measurement of viscoelasticity A platelet aggregation analysis program that causes a computer to analyze platelet aggregation based on the data. A platelet- inducing substance and a calcium salt are added to a platelet-containing sample and stirred, and after stirring, measurement data of electrical characteristics and/or viscoelasticity obtained from the platelet-containing sample, and the platelet-inducing substance in the platelet-containing sample. added which do rather than calcium salts, are stirred at least one is added from among the group consisting of saline and buffer, after stirring, the platelet electrical characteristics obtained from a sample containing and / or measurement of viscoelasticity The program for analyzing platelet aggregation ability according to claim 7 , which comprises comparing with data. A biological sample holding unit for holding a platelet-containing sample,
A drug supply unit that supplies a platelet-inducing substance and/or a calcium salt to the platelet-containing sample,
A stirring mechanism for stirring the platelet-containing sample to aggregate platelets activated by the platelet-inducing substance ,
A measurement unit for measuring the electrical characteristics of the platelet-containing sample in a non-stirred state ;
A time control unit for controlling the stirring time for stirring the platelet-containing sample,
A drive mechanism for controlling the drive operation of the stirring mechanism,
And an analysis unit that analyzes a platelet contribution unit in blood coagulation based on the electrical characteristics measured by the measurement unit ,
A platelet- inducing substance and a calcium salt are added to a platelet-containing sample and stirred, and after stirring, measurement data of electrical characteristics and/or viscoelasticity obtained from the platelet-containing sample, and the platelet-inducing substance in the platelet-containing sample. added which do rather than calcium salts, are stirred at least one is added from among the group consisting of saline and buffer, after stirring, the platelet electrical characteristics obtained from a sample containing and / or measurement of viscoelasticity A computer is made to analyze the platelet aggregating ability based on the data, a platelet aggregating ability analyzing apparatus incorporating a computer equipped with a program for platelet aggregating ability, and a display device for displaying the result of the analysis, Platelet aggregation analysis system.